Members of the CD28 family (CD28 and ICOS) play key roles in regulating T cell activation in response to antigen. For this reason they are attractive targets of immunotherapy regimes which aim to either attenuate the T cell responses to prevent allergic reactions, rejection of transplanted tissue and autoimmune disease, or augment responses against poorly immunogenic tumor or viral antigens. CD28 and ICOS provide costimulatory signals that allow a T cell to secrete effector cytokines but only CD28 costimulation protects a cell from apoptosis and permits long-term expansion. While much is known about the effects of the CD28 family on T cell activation, considerably less is understood about the signaling pathways that control these diverse functional outcomes. For instance, several groups have observed that CD28 costimulation enhances the expression of Bcl-xL and IL-2 using distinct mechanisms but the pathways that lead to this distinct regulation are unknown. Our approach is to utilize the structural similarity of the CD28 family to create a series of loss of function (LOF) and gain of function (GOF) mutant that will allow us to probe the mechanisms by which CD28 and ICOS alter T cell activation in primary human T cells. In Specific Aim 1 we will define the motifs responsible for the enhanced regulation of Bcl-xL and IL-2 and use these LOF and GOF mutants to characterize these distinct CD28 mediated signals. Aim 2 will address the question of how many signaling motifs are operational within the ICOS cytoplasmic tail and define the pathways that are activated by these motifs. Finally, we will develop a cell based model to validate our finding using a cell based model that uses the natural ligands to activate the T cells and permits ligand/receptor movement with the respective cell membranes. The data generated from these studies will better define the relationship between antigen dependent and independent signals and enable new strategies to modulate T cell activation for therapeutic applications.